Team:Groningen/protocols/Transformation EC

E. coli transformation protocol

Materials:

• LB plates with selection markers (antibiotics, inducers,…)
• LB broth
• Eppendorf tubes (preferably 2 ml)
• Spreaders
• DNA to be transformed

Steps:

1. Prepare plates with the correct antibiotic selection marker.
2. Prechill the eppendorf tubes on ice.
3. Add 10μl ligation reaction to a sterile tube.
4. Remove a tube of frozen competent E. coli (DH5α) cells from the storage (-80°C freezer) and thaw it on ice (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent cells are extremely fragile.
5. Carefully transfer 50μl of cells into each tube prepared in Step 2.
6. Add 10μl ligation reaction to the competent cells.
7. Gently flick the tubes to mix and place them on ice for 30 minutes.
8. Heat-shock the cells for 60 seconds in a water bath at 42°C (do not shake and make sure the content of the tube is all at the bottom).
9. Immediately return the tubes to ice for 2 minutes.
10. Add 1ml LB broth (RT) to the reaction mixture.
11. Incubate for at 37°C 1 hour with shaking (~150rpm).
12. Centrifuge the tubes at 14000 rpm for 1min.
13. Empty the tube until about 200μl supernatant is left.
14. Resuspend the pellet in the supernatant.
15. Plate 200μl of each transformation culture on LB agar plates with an ampicillin resistant marker.
16. Incubate the plates overnight (16–24 hours) at 37°C.
17. Afterwards plates can be stored at 4°C.